SOLID PHASE PEPTIDE SYNTHESIS

THE SOLID PHASE PEPTIDE SYNTHESIS IS SLIGHTLY DIFFRENT FROM PEPTIDE SYNTHESIS WHICH IS DISCUSSED HERE, ITS SYNTHESIS WITH STRUCTURE ANS BASICS ARE DISCUSSED WHICH WILL BE VERY USEFUL FOR READERS.

1. Solid phase peptide synthesis
Part I
Applicationsof Boc/Bzl strategy

2. Outline
Why peptide synthesis is necessary
Solid phase peptide synthesis
(idea, comparison with the synthesis in solution);
Resins;
Protecting groups;
Synthetic protocol;
Monitoring;
Cleavage procedures;
Side reactions;

3. Applications of
synthetic peptides
Immune peptides:
synthetic antigens;
vaccines
diagnostic tools
immunostimulator peptides;
muramyl dipeptide
tuftsin derivatives
Hormones:
oxytocin
vasopressin
insulin
somatostatin
GnRH
etc.
Neuropeptides:
substance P
cholecystokinin
neurotensin
Antibiotics:
tachikinin
gramicidine S
Toxins:
conotoxins
spider toxins
snake toxins
ionchanel blockers
Enzymes and
enzyme inhibitors:
Ribonuclease A
Carriers:
templates
miniproteins
Peptides
for structural studies:
turn mimicking cyclic peptides
Transporter peptides:
penetratin
oligoarginine
HIV-Tat protein

4. Why chemists are needed?
Gene expressionis very popular, relatively easy and cheap method:
it is good for long linear peptides or proteins containing L-amino acids.
However:
no D-amino acids
no unnatural amino acids
no post translation (Hyp, Pyr, glyco-and phosphopeptides)
no branches
no cyclic peptides
no fluorescent or isotop labeling
Peptides as drugs: there are not too many, because of the price and their
fast biodegradation.
“Peptideshaveandwillcontinuetobeimportantsourcesofleadcompoundsinmany
drugdiscoveryprograms.However,duetotheirgenerallypoorpharmacokinetic
propertiesandhydrolyticinstability,naturalpeptidestructuresareusually
substitutedwithmimicsoftheactualpeptideconstuction.”

5. Peptidek mint gyógyszerek ?
A peptidekhez, fehérjékhez számos biológiai és élettani funkció kapcsolható.
Ezért a '60-as évektől a jövő gyógyszereinek gondolták.
Előnyök: nagy specifitás, magas aktivitás, viszonylag kis dózis, kicsi toxicitás,
kevés mellékhatás.
Hátrány: gyors lebomlás, magas költségek.
2000-ben a világ gyógyszeriparának kb. 265 milliárd USD bevételéből
28 milliárd USD a peptidek és fehérjék bevételéből származott.
Évente 35-40 új vegyület kerül gyógyszerként bevezetésre.
Ezek között a peptidek száma egyre növekszik.

6. Peptidek a piacon
Rekombináns fehérjék: >50 ~40 ~60
Monoklonális ellenanyagok: >20 >20 >45
Szintetikus peptidek: >40 >20 >60
piacon pre-regisztrációs fázis klinika-II
klinika-III
GnRH szuper-agonisták és antagonisták: tumor terápia
Szomatosztatin analógok: tumor terápia
ACE (angiotenzin konvertáló enzim) inhibitorok: vérnyomás szabályozás
HIV proteáz inhibítorok: AIDS ellen
Vazopresszin, Oxitocin, ACTH: hormonok
Kalcitoninok: oszteoporózis ellen
Immunstimuláló peptidek: szervezet védekező
képességének növelése
A. Loffet J. Peptide Science(2002) 8, 1-7.

7. PEPTIDE SYNTHESIS
Coupling of amino acids:
NH
2-CH(R)-COOH + NH
2-CH(R’)-COOH
-H
2O
NH
2-CH(R)-CO-NH-CH(R’)-COOH; NH
2-CH(R’)-CO-NH-CH(R)-COOH;
NH
2-CH(R)-CO-NH-CH(R)-COOH; NH
2-CH(R’)-CO-NH-CH(R’)-COOH;
+ oligomers and polymers with different composition
Protecting groups: amino-; carboxyl-; side chain protecting groups
X-NH-CH(R)-COOH + NH
2-CH(R’)-COOY
-H
2O
X-NH-CH(R)-CO-NH-CH(R’)-COOY;
Removal of the protecting groups together or selectively

8. Synthesis in solution Synthesis on resin (SPPS)
time consuming;
manual;
1.1-1.2 equiv amino acid derivatives
and coupling reagent for acylation;
side chain protecting groups for
Lys, Asp, Glu, (Cys);
coupling: less than 90% conversion;
purification after eachsteps;
large scale;
cheap.
fast;
synthesizer (or manual);
3-10 equiv amino acid derivatives
and coupling reagents for acylation;
side chain protecting groups for
all functional groups
coupling: over 99.5% conversion;
purification at the end;
rather small scale;
expensive.
Synthesis of a
h-ACTH (1-39) in solution took months for several chemists;
A 39-mer peptide by SPPS 2 days, 1 day cleavage, 1-2 days purification,
1 week altogether for 1 chemist.

9. SOLID PHASE PEPTIDE SYNTHESIS
Bruce Merrifield published in 1963
Nobel Prize in Chemistry in 1984
The idea:
T
P
AA
1 RX
anchoring
T
P
AA
1R
deprotection
P
AA
1R
T
P
AA
2
coupling (-H
2O)
T
P
AA
2
P
AA
1R

10. T
P
AA
2
P
AA
1R
deprotection
coupling
repetitive
cycle
T
P
AA
n
P
AA
2
P
AA
1R
AA
n
AA
2 AA
1
cleavage
+
final deprotection

11. STRATEGIES
Boc/Bzl:
CH
2
NHCH
CH
2
C
O
O
C
O
NHCH
2
C
O
NHCHC
O
O
CH
2
O
CH
2
Cl
Cl
CH
2
RCH
3
CO
CH
3
CH
3
C
O
TFA
HF
HF
Boc-Asp(OBzl)-Gly-Tyr(2,6-Cl
2Bzl)-Merrifield resin
Boc
Bzl
2,6-Cl
2Bzl

12. Fmoc/
t
Bu:
NH
C
CH
2
OCNHCH
CH
2
C
O
O
C
O
H
C
CH
3
CH
3CH
3
NHCHC
O
O
CH
2
CH
2
OCH
2
O
O
C
CH
3
CH
3
CH
3
C
R
Fmoc
tert-butyl
..
piperidine
TFA
Wang-resin
Fmoc-Asp(O
t
Bu)-Tyr(
t
Bu)-Wang resin

13. RESINS
Can be functionalised;
Chemical stability(it must be inerttoallapplied chemicals);
Mechanical stability(it shouldn’tbrake under stirring);
It must swell extensively in the solvents used for the synthesis;
Peptide-resin bond should be stable during the synthesis;
Peptide-resin bond can be cleaved effectively at the end of the synthesis;
The basic of the most common used resins:
polystyrene-1,4-divinylbenzene (1-2%) copolymer
+
polymerisation

14. Type of resins for Boc-chemistry
CH
2Cl P
Merrifield (chloromethyl) resin
NH
3
CH
2NH
2 P
Aminomethyl resin
Starting resin for the synthesis
of many other resinsCH
2
OH CH
2
C
O
OH
CH
2
OH CH
2
C
O
NHCH
2 P
PAM resin (phenyl-acetamidomethyl)
p-hydroxymethyl-phenyl-
acetic acid (handle)
+ DIC

15. CH
2Cl P
Boc-Aaa-O
-
Cs
+
DMF, 50
o
C, 48h
CH
2
OH CH
2
C
O
NHCH
2 P
CH
2
OBoc-Aaa- CH
2
C
O
NHCH
2 P
Boc-Aaa-OH
DIC + 10%DMAP RT
DCM-DMF (1:3) 1h
Peptide-PAM resin bond is more TFA stable than Peptide-Merrifield resin bond.
The final cleavage results in peptides with carboxyl (COOH) group
at the C-terminus.
Attachment of the first amino acid to Merrifield and PAM resins
CH
2
OBoc-Aaa- CH
2
C
O
OH
CH
2
NH
2 PDIC +
CH
2Boc-Aaa-O P

16. The final cleavage results in peptides with carboxamide (CONH
2) group at
the C-terminus.
CHNH
2
P
Boc-Aaa-OH
DCC/HOBt
CHNH PCCH(R)NH
O
Boc
Benzhydrylamine resin (BHA):
CHNH
2
P
Boc-Aaa-OH
DCC/HOBt
CHNH PCCH(R)NH
O
Boc
4-Methyl-benzhydrylamine resin (MBHA):
CH
3
CH
3
too stable underacid cleavage conditions
(only; HF!)

17. Coupling capacity of the resin
Preloaded resins are commercially available
(coupling capacity, writtenon the box is expressedin mmol/g);
BHA and MBHA resin(the NH
2content is given on the box)
Attachment of the first amino acid is usually performed with
100% yield;the resincapacity will be the same;
Coupling of p-hydroxymethyl-phenoxy acetic acid containing Boc-
amino acid to aminomethyl-resinrepresentsa similar situation;
Attachment of Boc-amino acid derivative to Merrifield or PAM-
resin (Kjeldahl N analysis, elemental analysis, amino acid analysis
or titration by pycric acid after Boc-removal: colour test)
Kjeldahl N analysis:
cc. H
2SO
4for 24 hrs
add base
NH
3destillation into water
titration with 4mM H
2SO
4
calculation of % N to mmol/g
Lys (2N), His (3N), Arg (4N)
Amino acid analysis:
6M HCl in an evacuated and
stopped tube (hydrolysis)
heating at 110
o
C for 24 hrs
evaporation, neutralisation
amino acid analysis
(quantitative)

18. Applied side chain protecting groups in Boc-chemistry
benzyl (Bzl)CH2-OH (Ser, Thr, Tyr)
Side chain functional group protecting group name (abbreviation)
HF
intramolecular intermolecular
NHCHC
O
CH
2
OR
NHCHC
O
CH
2
O
H R
+
NHCHC
O
CH
2
O
H
R
+
NHCHC
O
CH
2
OH
R
R
+
can be caught by scavangers
However in case of Tyr:
20-100% side product!
The side reaction
cannotbe avoided
by using scavangers.
Mw: +90.05

19. Side chain functional group protecting group name (abbreviation)
2,6-dichlorobenzyl
(2,6-di-Cl-Bzl)CH2-OH (Tyr)
Cl
Cl
OCH2
Br
C
O
2-bromobenzyl-
oxycarbonyl
(2-Br-Z)
Bu
t
< 2-Br-Z < cHex < 2,6-di-Cl-Bzl < Bzl
0,05% 0,1% 0,5% 5,0% 20%
Electrophilicity order of carbocations:
(amount of 3-alkyltyrosine in the peptide)
cyclohexyl
(cHex)
It is not commercially available
O N acyl shift;
donotkeep the peptide
without a-NH protection
for long time !

20. --C----C---C------C---
Acm Acm
S S
--C----C---C------C---
S S
S S
Side chain functional group protecting group name (abbreviation)
4-methylbenzyl
(Meb)
-SH (Cys) CH2 CH3
CH2 CH3O
4-methoxylbenzyl
(Mob)
Stability vs TFA is
not good enough;
not for longer peptides!
CH2NHC
O
CH3
acetamidomethyl
(Acm)
For selective deprotection
--C----C---C------C---
Acm Acm
Meb Meb
HF
--C----C---C------C---
Acm Acm
SH SH
air
oxidation
I
2or Tl(tfa)
3
Hg(II)-or Ag(I)-salt !
Eg.

21. Side chain functional group protecting group name (abbreviation)
3-nitro-2-pyridinesulphenyl
(Npys)
-SH (Cys)
S
N
NO
2
For the synthesis of asymmetrical disulfide dimers
stable in the presence ofacids
cleavable by bases
or thiols
Not for Fmoc-chemistry!
Eg.
--------C-------R
Npys
HF
--------C-------OH
Npys
Bzl
---C-----NH
2
SH
+ in acidic buffer
(pH 5-6)
---C-----NH
2
S
--------C-------OH
S--------C-------OH
S
--------C-------OH
S
---C-----NH
2
S
---C-----NH
2
S
at pH > 7
Neutral or basic
condition is not
appropriatefor
asymmetrical
disulfide bond
formation!

22. O
OCH2
Cl
C
2-chlorobenzyl-
oxycarbonyl
(2-Cl-Z)
Side chain functional group protecting group name (abbreviation)
O
OCH2C
benzyloxycarbonyl
(Z)
e
NH
2
(Lys)
Z is not stable enough
inTFA;
branches in the peptide !
w
COOH (Asp, Glu)
OCH2
O
benzyl(ester)
(OBzl)
cyclohexyl(ester)
(OcHex)
OBzl is not stable enough
inTFA;
lead to ringclosure
side reaction !

23. Succinimide ring formation (Asp):
-Asp-Gly-
NHCHC
O
NHCH
2
C
O
CH
2
C
OBzl
O
-Asu-Gly-
NHCH
N
CH
2
C
C
O
CH
2
O
C
O
NHCH
-BzlOH
Asp-X; X =Gly,Arg,Ala, Ser, Asx
NHCHC
O
NHCH
2
C
O
CH
2
C
OH
O
~30%
NHCH
CH
2
C
C
O
O
NHCH
NCH
2
C
O
OH
~70%
a-Asp-peptide b-Asp-peptide
+H
2O
+H
2O
Molecular weight is the same in both cases; HPLC separation of isomers in case
of small peptides; enzymatic degradation amino acid analysis.

24. NH
2
CHC
O
NHCHC
O
CH
2
C
OBzl
O
CH
2
R
NHCHC
O
NHCHC
O
CH
2
CO
CH
2
R
-BzlOH
Pyroglutamic acid formation at the N-terminal of the peptide (Glu):
M= M
calc-18
NH
2
CHC
O
NHCHC
O
CH
2
C
NH
2
O
CH
2
R
NHCHC
O
NHCHC
O
CH
2
CO
CH
2
R-NH
3
M= M
calc-17
Don’t prepare peptides containing Gln at the N-terminus
They are not presentin the nature!
QXNAD: X= K(21%), Arg, His(18%), Ala(11%), Leu(8%), Tyr(7%), Asp(4%), Glu(2%)
After 48h at pH 7, 37
o
C: His(51%), Arg(32%), Leu(19%), Tyr(22%), Asp(21%)
Acidic pH, elevated temperature, X= D-Aaa; increase the Glp content

25. O
Boc-NHCHC
O
NHCHC
O
CH
2
C
NH
O
CH
2
R
Side chain functional group protecting group name (abbreviation)
w
CONH
2(Asn, Gln)
O
xantyl (Xan)
33%TFA/DCM deBoc, deXan
Why do we use Xan protecting group?
not necessary, but;
increase the solubility of
Boc-Gln-OH and Boc-Asn-OH,
eliminate the nitryl formation.
NH
2
CHC
O
NHCHC
O
CH
2
C
NH
2
O
CH
2
R
NHCHC
O
OH
CH
2
C
N CH
2
DCC
Boc
O
NHCHCOH
CH
2
C
NH
2
O
CH
2
Boc

26. Side chain functional group protecting group name (abbreviation)
NO
2
Protection of
t
N prevents
the alkylation or acylation
of imidazol ring, but not
the epimerisation of His;
Protection of
p
N prevents
also the epimerisation.
N N
H
(His)
p
t
imidazol group
SO2 CH3
p-toluolsulfonyl
or tosyl (Tos) (t)
Itistoosensitiveinthepresenceofweakacids
likeHOBt,however,itistoostableinHF.
NO
2
dinitrophenyl
(Dnp) (t)
Special cleavage procedure:
thiophenol:DIEA:DMF = 3:3:4 (V/V/V)
several times; long reactiontime (yellow colour)
Boc-Aaa
1-Aaa
2(Bzl)-His(Dnp)-....-Resin
Boc-Aaa
1-Aaa
2(Bzl)-His-....-Resin
NH
2
-Aaa
1-Aaa
2(Bzl)-His-....-Resin NH
2
-Aaa
1-Aaa
2-His-....-OH
HF
TFA
thiophenol

27. However, Bom must notbe usedin case of peptides containing Cys at the N-terminus:
Side chain functional group protecting group name (abbreviation)
OCH
2CH
2
N N
H
(His)
p
t
imidazol group
benzyloxymethyl
(Bom) (p)
Cleavage of Bom results in Bzl
+
and CH
2O;
Formaldehyde can react with nucleophiles:
H
2C=O +
e
NH
2-Q H
2C=N-Q (Schiff base)
H
+
H
2C=O + OH-R H
2C-OH
O-R
H
2C-O-R
O-R
hemiacetale acetaleWork up the peptide as soon as possible !
HNH-CH-CO---
CH
2
SH
H
2C=O
-H
2O
NH-CH-CO---
CH
2
S
H
2C
thiazolidine-4-charboxylic acid
(thioproline)
M=M
calc+12
Use Cys as scavanger under the cleavage
condition to catch the formaldehyde !

28. Racemisation
C
R
O
N
H
C
R’
H
C
ActO
O
Acyl-L-Aaa-OAct
B:
-ActOH
5(4H)-oxazolone
C
R
O
N
C
R’
H
C
O
-H
+
C
R
O
N
C:
-
R’
C
O
C
R
O
N
CR’
C
O:
-
C
R
O
N
CR’
C
O:
-
Pseudo aromatic
system
+H
+
C
R
O
N
C
R’
H
C
O
DL
-H
+
H-L-Aaa-OY
DL and LL dipeptide derivatives
Direct proton withdrawn
or oxazolone formation;
No racemisation in case
of Gly and Pro (through
oxazolone);
No racemisation with
uretane type proecting
groups (Boc, Fmoc);
His: proton transfer

29. 2,3,6-trimethyl-
benzenesulfonyl or
mesitelenesulfonyl
(Mts)
Side chain functional group protecting group name (abbreviation)
-NH-C-NH
2
NH
CH
3
S
O
O
p-toluolsulfonyl
or tosyl (Tos)
CH
3
S
O
O
CH
3
CH
3
CH
3
O
4-methoxy-2,3,6-
trimethylbenzene-
sulfonyl (Mtr)
guanidino group
CH
3
S
O
O
CH
3
CH
3
Lability in acids:
Mtr > Mts > Tos
Cleavage:
Tosonly in HF
(TFMSA or TMSOTf at RT;
not recommended)
Mts all of them
MtrTFA forextended time
(it was used inFmoc-chem.)
In the synthesis of oligo-Arg (cellpenetrating peptide) use Mts or Mtr protection
Application of sulfonyl type protecting groups: the protection of Trp is suggested
(Arg)

30. Side chain functional group protecting group name (abbreviation)
indole
H-C
O
formyl (For)
Special cleavage is necessary:
20% piperidine/DMF before HF cleavage
or low-high HF cleavage procedure
OC
O
cyclohexyloxy-
carbonyl (Hoc)
Side reaction under
acidic condition:
oxidation
alkylation
Oxidation of Trp results in oxyindolyl and kynureninyl derivatives; pink colour
Alkylation by tert-butyl cation resulted under TFA cleavage of Boc-group
N
H
*
*
*
*
M
1= M
calc+ 56.06
M
2= M
calc+ 112.12
M
3= M
calc+ 168.18
In case of the application of Trp without
any protection, add anisole and indole as
scavangers to the TFA cleavage mixture
(10mL TFA : 0.3mL anisole : 0.1g indole) !
N
H
(Trp)

31. Side chain functional group protecting group name (abbreviation)
-CH
2
-CH
2
-S-CH
3
sulfide (Met)
-CH
2
-CH
2
-S-CH
3
O
sulfoxide (O)
Side reaction under
acidic conditions:
oxidation
alkylation
Oxidation of Met results in its sulfoxide form.
Alkylation by benzyl or tert-butyl cation:
-CH
2
-CH
2
-S-CH
3
-CH
2
-CH
2
-S-CH
3
CH
2
+
-CH
2
-CH
2
-S-CH
2
-CH
3
OH
M = M
calc + 76.03 in case of Bzl
M = M
calc + 42.05 in case of tBu
In case of the application of Met without any protection, add anisole and
Met as scavangers as well as DTT as reductive agent to the TFA cleavage
mixture (10mL TFA : 0.3mL anisole : 0.1g Met : 0.1g DTT) !
Removal:N-methylmercaptoacetamide, low-high HF,
NH
4I, TMSOBr+thioanisole

32. Synthetic protocol of Boc-strategy
1)Wash the resin 3x with DCM; 0.5-1.0 min each
2)Cleavage of Boc protection with 33%TFA/DCM; 2+20min
3)Wash the resin 5x with DCM; 0.5-1.0 min each
4)(Shrinking the resin with 25%dioxan/DCM)
5)Neutralisation 3-4x with 5-10%DIEA/DCM; 1 min each
6)Wash the resin 4x with DCM; 0.5-1.0 min each
7)Coupling: Boc-amino acid derivative-DCC-HOBt in DCM-DMF *
(3 equiv each calculated to the resin capacity); 60 min
8)Wash the resin 2x with DMF; 0.5-1.0 min each
9)Wash the resin 2x with DCM; 0.5-1.0 min each
10)Ninhydrin monitoring **
(-) yellow
(+) blue
*The ratio of DCM and DMF depends on the solubility of the amino acid
derivatives; DCM-DMF = 4:1 or 2:1 (V/V) in most cases.
However, in case of Arg, Gln, Asn DCM:DMF 1:4 or 1:2 (V/V) is prefered.
**When coupling is carried out toPro, the ninhydrin assay can’t be used.
Application of isatin test or bromophenol blue test is necessary.

33. When might be double coupling necessary
Incorporation of the 10-15th amino acids;
Attachment to Pro;
Coupling of amino acids containing a branch on b-C atom (Val, Ile, Thr);
Attachment to these amino acids;
Coupling of Arg or attachment to Arg;
Attachment to e-amino group of Lys (synthesis of branched peptides).
Influence on the efficacy of the coupling:
Solvent: change DCM-DMF to NMP (N-methyl-pyrolidone)
Coupling reagent: change DCC/HOBt to BOP, HBTU or HATU
Application of these expensive reagents is suggested for the third coupling.
If the nynhidrine test is still blue make acetylation to block the
unreacted amino groups (acetic anhydride and DIEA in DMF).

34. Isatin test:
3% isatin + 5% Boc-Phe-OH
dissolved in benzylalcohol
+ ninhydrin test solution
Colour of resin is red to black
Ninhydrin monitoring
O
OH
OH
O
2
+ NH
2-R
O
OH
N
O
O
blue l(570nm)
O
-
OH
N
+
In case of Pro:
There is no difference
between the colour of
ninhydrine and the product
yellow
Test solutions:
40 g phenol in 10 mL abs. EtOH
65 mg KCN in 100 mL d.i. water
(take 2 mL and dilute with 98 mL pyridine)
2.5 g ninhydrin in 50 mL abs. EtOH
NH
O
O

35. Monitoring with bromophenol blue
3’,3”,5’,5”-tetrabromophenolsulfonphtalein:
Br
Br
O
Br
Br
OH
S
O
O
OH
NH
2-R
Br
Br
O
Br
Br
OH
S
O
O
O
-
HNH
2-R
+
l
max= 429 nm l
max= 600 nm
the change of the colour is because of salt formation (non covalent bond)
highly sensitive
the coupling can be followed (blue green yellow)
application of amine-free DMF is necessary
1% BB solution in dimethylacetamide; 2-3 drops to the reaction mixture
25 mL 0.04M solution for analysis
available for checking the coupling to Pro

36. Diketopiperazine formation:
Synthesis cycle:Deprotectionwith 100% TFA 2x1 min
Washwith DMF 30 sec flow wash
Coupling: Boc-Aaa-derivative:DIC:HOBt (4 equiv each)
+ 1.5 equiv DIEA (calculated to the resin capacity
Washwith DMF 30 sec flow wash
In situ neutralisation
Apply this method when there is a danger of:
diketopiperazine formation; Pro containing dipeptide
pyroglutamic acid formation; Glu(Bzl), Gln on the N-terminus
”difficult” sequence, aggregation; a-helical or b-sheet structure
CH
2C O P
O
R
1-CH
NH
C
O
CH-R
2
NH
2
C
O
R
1-CH
NHC
O
NH
CH-R
2
CH
2 PHO
+
Pro-Pro
Pro-Gly
Gly-Pro
D-Aaa-Pro
Pro-D-Aaa
cis-peptide bonds
(Preactivation is necessary) CF
3COO
-+
NH
3-CHRCO

37. Boc cleavage flow chart
Does the peptide
contain His(Dnp)?
yes
no
Remove Dnp Does the peptide
contain N-terminalBoc group?
yes
no
Remove Boc Is the peptide (protecting groups)
compatible with HF, TMSOTf, TFMSA?
HF
Does the peptide
contain Trp(For)?
yes no
Deformylate
Trp(For) or
”Low-high” HF cleavage
HF cleavage
TMSOTf
TFMSA
Does the peptide
contain Trp(For) or Met(O)?
TMSOTf cleavage
no
yes
”Low-high” TFMSA cleavage
Standard TFMSA cleavage

38. Why is it necessary to remove Boc-group before
cleavage with strong acids?
tert-butyl cation is a very effective alkylating agent;
long cleavage time, high cation concentration;
the best scavanger to trap the tert-butyl cation is water;
however water can’t be used with strong acids because of splitting
of peptide bonds;
there are some special side reactions, eg. in case of peptides containing
Met at the C-terminal (homoserine lactone formation);
CH
3
S
NH
O
O
R
HF
CH
3
S
NH
OH
O
+
O
NH
O
M = M
calc-47.0

39. Problems withthe cleavage procedures
HF : needs a special teflon instrument. However all the applied
protecting groups can be cleaved. Cleavage time is 45-60 min at 0
o
C,
but in case of Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) 90 min is
recommended. Anisole, p-cresol and DTT as scavangers are used.
TMSOTf : 1 M TMSOTf-thioanisole/TFA solution in the presence of
m-cresol and EDT at 0
o
C for 120 min.
Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) and BHA resin
are not cleavableundertheseconditions.
TFMSA : 10% TFMSA-10% thioanisole in TFA at RT for 1.5-2hrs.
EDT and m-cresol arerecommended as scavangers.
Arg(Tos), Cys(Meb), Asp(OcHex), Glu(OcHex) and BHA resin
are not compatiblewith this method.
More side reactionsthan in case of TMSOTf.
Desalting is necessary at the end.

40. Cresol is prefered in case of Glu:
OCH
3
NH
O
O
NH
O
Don’t use indole as scavanger for Trp instrong acids
M = M
calc+ 90.05
M = M
calc+ 117.1
N
H
H
N
H
R-CH
2
H
H
Indole dimerisation can occur also
in case of peptides containing Trp
at the N-terminus resultingin dimer
peptide connected through indole rings.
Asp-Pro bond mightbe cleaved under acidic condition
Use dried materials and equipments !

41. 2-mercaptopyridine (10 equiv.) was suggested to prevent Met(O)
formation or Met(O) reduction under HF cleavage. However it
decreases the acidity of HF, so some protecting groups (eg. Tos)
can’t be removed effectively. Add Met and DTT to eliminate
Met(O) formation under HF cleavage.
N-O acyl shift in case of Ser or Thr
O
NH
R
O
OH
NH
R=H (Ser), CH
3
(Thr)
NH
R
O
O
OH
NH
NH
3
R
O
O
O
NH+
This reaction can be reversed by
either neutralizing with NH
4OH or
relyophilisation from 5% NH
4HCO
3

42. ”Pull-push” mechanism in the presence of thioanisole
CH
2
OCH
2
R
SiMe
3
-O
3
S-CF
3
SCH
3
OCH
2
R
SiMe
3 CH
2
SCH
3+
+
CF
3
SO
3
-
H
2
O
(NH
4
F)
OHCH
2
R
m-cresol
HO
CH
3HO-SiMe
3
+
CH
2
SCH
3
CF
3
SO
3
H
+
Thioanisole = reversible scavanger
Cresol = irreversible scavanger
Don’t use reversible scavanger alone !

43. ”Low-high” HF cleavage
Standard HF cleavage (S
N1):
10 mL HF
0.5-1.0 g scavanger (anisole, p-cresol)
0.1 g DTT or 0.5-1.0 mL EDT or DMS
as reducing agent
45-90 min dependingon the protecting
groups
from -15
o
C to 0
o
C, dependingon the
sequence (side reactions)
”Low-high” HF cleavage (S
N2+ S
N1):
First step (low);there isno carbocation
2.5 mL HF
0.75 g p-cresol + 0.25 g p-thiocresol
6.5 mL DMS
2-3 hrs
0
o
C
evaporation of HF and DMS
(it takes quite a long time)
Second step (high):
standard HF cleavage
new HF and scavangers
45 min
0
o
C
Low HF:
Met(O) Met
Trp(For) Trp
100% cleavable:
Arg(Mtr), Arg(Mts), Asp(OBzl)Glu(OBzl),
Lys(Z), Lys(ClZ), Ser(Bzl), Thr(Bzl),
Tyr(BrZ), Tyr(Bzl), Merrifield resin
Cys(Mob), Tyr(2,6-Cl2Bzl), PAM resin (<80-85%)
His(Bom) (<60%)
The other protecting groups
can be cleaved just by high
HF procedure.
TFMSA (15%)-DMS(30%)-TFA(55%)

44. Synthesis of ”head to tail” type cyclic peptides
on resin
Application of oxim resin:
CNO
2
P
N
HO
Boc-Aaa(X)-OH
+DCC/DCM
p-nitrobenzophenone oxim resin
CNO
2
P
N
Boc-Aaa-O
The peptide-resin bond is stable
in acids, but cleavable by amines.
Compatible just with Boc chemistry.
However in situ neutralisation is
necessary.
CNO
2
P
N
NH
2
-PEPTIDE-O
c(PEPTIDE)
CNO
2
P
N
Boc-PEPTIDE-O
NH
2
-Aaa(X)-OY
Boc-PEPTIDE-Aaa-OY
Synthesis of cyclic peptides
and protected peptide fragments

45. Synthesis of cyclopeptides
What is the reason of cyclopeptides synthesis?
1. Natural compounds: antibiotics, hormones, toxins, enzymes,
immunoglobulines, depsipeptides, etc.
gramicidine S (antibiotic): Val-Orn-Leu-D-Phe-Pro
Pro-D-Phe-Leu-Orn-Val
somatostatine (hormone):
H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
a-conotoxin GI (toxin):
H-Glu-Cys-Cys-Asn-Pro-Ala-Cys-Gly-Arg-His-Tyr-Ser-Cys-NH
2

46. phalloidine (toxin in mushrooms):
N
H
S
NH
CO
CO
CR
2
NH
CO
CR
1
NHO
CO
NH
ONH
CR
3
CO
NH
CR
4
CO
2. Increasing or change the biological activity of the cyclic peptides:
eg. Somatostatine derivative with high antitumour activity;
H-D-Phe-Cys-Tyr-D-Trp-Lys-Cys-Thr-NH
2

47. 3. Structurestabilization:
eg. for improvement of the hormone-receptor interaction (increased
selectivity);
Leu-enkephaline: H-Tyr-Gly-Gly-Phe-Leu-OH
Cyclic derivative: H-Tyr-Dab-Pro-Phe-Leu
Dab = a,g-diaminobutiric acid;
g
NH
2
-CH
2
-CH
2
-CH
2
-COOH
a
NH
2
4. Increasedenzyme stability:
GnRH-III (antitumour activity):
Pyr-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH
2
Pyr-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH
2
Pyr = pyroglutamic acid
Selective for
m-receptor

48. 5. Study of the structuralelements:
c(b-Ala-Ala-b-Ala-Pro) has g-turn conformation
6. Templates: for eg. synthesis of miniproteins
G
K
C
K
P
P
K
C
K
G
S
S
The template contains amide bonds in the cycle
and it is fixed by disulfide cross-linkage.
Selective protection of Lys residues allows
attachment of 4 different peptide chains.
Arrangement of cyclic peptides
homodetic heterodetic
only amide bonds in the cycle
disulfide bridge, thioether bond
lacton, ether, oxime thiazolidine bond